The Old World and the New World alphaviruses represent two groups of geographically isolated viruses, which independently evolved for almost 3,000 years. Accordingly, they accumulated strong differences in the nonstructural and structural genes and cause different diseases in vertebrate hosts. The New World alphaviruses, which include Venezuelan (VEEV), eastern and western equine encephalitis viruses, are continuously circulating in the Central, South and North Americas and have the ability to cause encephalitis with frequent fatal outcomes in humans and horses. We have recently demonstrated that the Old World and the New World alphaviruses differ not only in the severity of the disease, but also in the molecular mechanisms of virus-host interactions, particularly by interfering with the cellular antiviral response. Thus, our knowledge about replication of the less pathogenic, Old World alphaviruses cannot be directly applied to the encephalitogenic New World viruses, as they have accumulated strong differences in their replication mechanisms. In the preliminary studies, we found that the nonstructural nsP3 proteins of geographically isolated viruses, VEEV and Sindbis, form distinct intracellular complexes and interact with different host factors. We have also found that the carboxy terminal fragment of VEEV nsP3 is critical for virus replication. nsP3 is the least studied viral protein, and it[unreadable]s functions in the viral life cycle have not yet been defined. No information about VEEV nsP3 in particular is available to date. In this project, we will perform extensive characterization of the structures and functions of different VEEV nsP3-containing complexes, and define the roles of the hypervariable fragment and nsP3 phosphorylation in viral replication. Understanding the role of VEEV nsP3 in virus replication will lead to development of new therapeutic means and the next generation of vaccines against highly pathogenic alphaviruses.